Summary This application focuses on upgrading the E. coli-based cell-free expression system and on optimizing the synergy between cell-free and NMR to screen and evaluate for structural studies by NMR and X-ray methods a library of 3360 human integral membrane proteins. Currently there exists an enormous knowledge gap for membrane protein structures. Fewer than 20 human membrane protein structures are available in the Protein Data Bank. Membrane proteins mediate cellular interactions with the surrounding, and they are targeted by half of the commercially available drugs. Each new human membrane protein structure is, therefore, a potential target of rational structure-guided drug design, and the significance of new structures cannot be overstated. We propose to improve the existing CF expression system optimized in our lab for membrane proteins to enable proper disulfide bridge formation and a large-size protein expression, and to improve purification of CF-expressed targets designated for crystallization screening (Aim1). We will also optimize the NMR structure determination method for membrane proteins utilizing the synergy between CF and NMR. We will improve the combinatorial dual-isotope labeling (CDL) strategy developed in our lab and use CF to incorporate site-specific unnatural amino acids. We will also employ 19F and 13C-methyl labeling (Aim 2). We will use these technical improvements to comprehensively evaluate the proposed human membrane protein targets by testing their expression and their suitability for NMR and X- ray structural studies (Aim3).